Selective R-cadherin antagonist and methods

ABSTRACT

An isolated peptide useful as a selective antagonist of mammalian R-cadherin comprises 3 to 30 amino acid residues, three contiguous residues of the peptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaa is an amino acid residue selected from the group consisting of Asp, Asn, Glu, and Gln. Preferably Xaa is Asp or Asn. In one preferred embodiment the peptide is a cyclic peptide having 3 to 10 amino acid residues arranged in a ring. The selective R-cadherin antagonist peptides of the invention are useful for inhibiting the targeting of stem cells, such as endothelial precursor cells, to developing vasculature, for inhibiting R-cadherin mediated cellular adhesion, and for inhibiting retinal angiogenesis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional application forpatent Ser. No. 60/467,188, filed on May 1, 2003, which is incorporatedherein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with United States government support underGrants No. EY11254 and EY12598 from the National Institutes of Health.The United States government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to antagonists of mammalian cellularadhesion molecules. More specifically, the invention relates toselective peptide antagonists of mammalian R-cadherin (cadherin-4) andmethods of inhibiting cellular adhesion and retinal angiogenesistherewith.

BACKGROUND OF THE INVENTION

The cadherin family of molecules consists of transmembrane glycoproteinsthat function in calcium dependent, selective cell-cell interactions.These molecules play important roles during embryonic development andtissue morphogenesis by mediating cell recognition and cell sorting.Subfamilies of cadherins (classic cadherins, protocadherins,desmocollins, and other cadherin-related proteins) are characterized byvariable numbers of extracellular cadherin domains, a singletransmembrane segment, and a single cytoplasmic domain. The so-calledclassic cadherins (i.e., E, P, N, and R-cadherin) reportedly have fivetandemly repeated extracellular cadherin domains (EC1 -EC5) that engagein preferentially homophillic interactions, and a highly conservedcytoplasmic tail that mediates adhesion specific intracellularsignaling.

Cadherin mediated cell-cell adhesions occur as multiple cadherinmolecules expressed on adjacent cells interact, leading to the formationof adherens junctions. According to the cadherin zipper model proposedby Shapiro et al. Nature 1995; 374(6520):327-37, cadherin moleculeswithin the membrane of the same cell form tight parallel-strand dimers(i.e., so-called cis-dimers). As illustrated in FIG. 1, these cis-dimersthen bind to cadherin dimers expressed on adjacent cells (i.e.,trans-dimerization). Once a sufficient interaction is sustained,cadherin clustering can occur as more cadherin molecules are recruitedto the site, leading to interdigitation of molecules from the two-cellsurfaces. In this manner, relatively weak interactions can combine toform fairly strong cell-cell adhesions.

Upon initial cadherin adhesion, intracellular signals, transmittedthrough interactions of the cytoplasmic cadherin tails with α and βcatenin molecules, lead to reorganization of the cytoskeleton. Althoughthe association with actin filaments is not thought to affecthomophillic binding, their association helps hold the cadherin moleculesat the sites of interaction. In a symbiotic-type of relationship,cadherin clustering causes reorganization of the cytoskeleton andprovides points of attachment at the membrane, which are important forcellular changes that occur upon the formation of adherens junctions.Meanwhile, association with the cytoskeleton holds cadherins at thesites of interaction and helps recruit new cadherin molecules, thusmediating cadherin clustering. Calcium plays an important role as acofactor during cadherin clustering. Cadherin function is lost andmolecules become more susceptible to protease degradation in solutionswith insufficient concentrations of calcium ions (i.e., below about 2mM). This is due to the requirement of calcium to stabilize thestructure of cadherin molecules and provide proper orientation ofadjacent cadherin interfaces.

Although it is reported that each of the five extracellular classicalcadherin domains EC1 through EC5 plays an important role in mediatingcadherin dimerization, mutational analysis has suggested that themajority of residues that form the dimerization interface are foundwithin the N-terminal most cadherin domain (EC1) (Kitagawa, et al.,Biochem. Biophys. Res. Commun., 2000; 271(2):358-63.). However,relatively little is known about the mechanisms of specifichomodimerization between cadherin molecules.

Cadherins play a significant role during neuronal guidance anddevelopment of the central nervous system. Different subdivisions of thebrain are reportedly defined by differential expression of cadherintypes. Cadherins also play an important role in neural retinaldevelopment by specific expression within different regions of thedeveloping retina. For example, during embryonic chick retinadevelopment, B-cadherin is reportedly only found in Müller glia, whilecertain populations of bipolar cells express R-cadherin (also known ascadherin-4). Amacrine cells and a subset of ganglion cells expresscadherins 6B and 7. Within the inner plexiform layer of the retina,these same cadherins are only expressed in sublaminae associated withsynapsin-I positive nerve terminals, suggesting that distinct expressionprofiles contribute to synapse formation between specific subpopulationsof neurons during retina development. In the embryonic optic nerve,ganglion cell axon outgrowth is mediated by N-cadherin adhesion withR-cadherin-expressing glial cells.

Cadherin adhesion also plays a role in developmental retinalvascularization (Dorrell, et al. Invest. Ophthalmol. Vis. Sci. 2002;43(11):3500-10). Disruption of R-cadherin adhesion during formation ofthe superficial vascular plexus results in the loss of complex vascularinterconnections observed during normal vascular patterning. WhenR-cadherin adhesion is blocked during the subsequent formation of deepvascular layers, key guidance cues are lost causing the vessels tomigrate past the normal deep vascular plexuses and into thephotoreceptor layer.

The retina consists of well-defined layers of neuronal, glial, andvascular elements. Any disease or condition that alters the retinallayers even slightly, can lead to neuronal degeneration and significantvisual loss. The retinal degeneration mouse (rd/rd mouse) has beeninvestigated for over 70 years as a model for many diseases that lead tophotoreceptor cell death. In the rd/rd mouse, photoreceptor degenerationbegins during the first three weeks after birth as rod cells undergoapoptosis, attributed to a mutation in the β subunit of cGMP-dependentphosphodiesterase followed by cone photoreceptor death. Vascular atrophywithin the retina is temporally associated with photoreceptor cell deathin rd/rd mice as well. The vasculature appears to form in the normalcharacteristic fashion as three functional layers develop within thefirst three weeks. However, the vessels in the deep vascular layer beginto degenerate during the second week and by the end of the fourthpostnatal week, dramatic vascular reduction is observed as the deep andintermediate plexuses nearly completely disappear.

A population of hematopoietic stem cells resides in the normal adultcirculation and bone marrow, from which different sub-populations ofcells can differentiate along lineage positive (Lin⁺HSC) or lineagenegative (Lin⁻HSC) lineages. In addition, the present inventors havediscovered that endothelial precursor cells (EPCs), capable of formingblood vessels in vitro and in vivo, are present within the Lin⁻HSCsubpopulation. EPCs within the population of Lin⁻HSCs can target andstabilize the degenerating vasculature in rd/rd mice when injectedintravitrally to the eyes of the mice. Intravitreally injected Lin⁻HSCstarget astrocytes in the superficial vascular layer and are observedahead of the endogenous developing vascular network when injected atpostnatal day 2 (P2). As the endogenous vasculature reaches theperiphery of the retina, where the Lin⁻HSCs have targeted, the cells areincorporated into new blood vessels, forming functional mosaic vesselswith mixed populations of injected Lin⁻HSCs and endogenous endothelialcells. In addition, Lin⁻HSCs target the regions of deep and intermediatevascular layers of the retina before vascularization of these layers byendogenous endothelial cells had occurs. Incorporation of Lin⁻HSCsrescues the deep vasculature of rd/rd mice about 2 to about 3 fold overnormal and control Lin⁺HSC injected mice. In addition, rescue of thedeep vasculature prevents degradation of photoreceptors in the outernuclear layer of the retina. However, as there is no evidence to suggestthat these stem cells can undergo differentiation into retinal neuronsor glial cells, the mechanism of neuronal protection remains unknown.

The targeting of Lin⁻HSCs to the astrocytes and deep vascular regionsahead of natural developmental vascularization suggests that theLin⁻HSCs express cell-surface molecules that function in targeting,similar to targeting of the endogenous endothelial cells duringdevelopment. R-cadherin adhesion plays an important role in endothelialcell targeting to astrocytes and vascular plexuses during developmentalretinal angiogenesis.

R-cadherin has been identified and sequenced in a number of mammals.FIG. 2 depicts the amino acid sequence (SEQ ID NO: 1) of a human variantof R-cadherin preproprotein reported by Kitagawa et al. in theSWISS-PROT database as Accession No. NP 001785, version NP 001785.2,GI:14589893, the relevant disclosure of which is incorporated herein byreference. SEQ ID NO: 1 includes the amino acid sequence IDSMSGR (SEQ IDNO: 2) at positions 222-228.

FIG. 3 depicts the amino acid sequence (SEQ ID NO: 3) of another humanvariant of R-cadherin preproprotein reported by Tanihara et al. in theSWISS-PROT database as Accession No. P55283, version P55283, GI:1705542,the relevant disclosure of which is incorporated herein by reference.SEQ ID NO: 3 includes the amino acid sequence INSMSGR (SEQ ID NO: 4) atpositions 222-228.

FIG. 4 depicts the amino acid sequence (SEQ ID NO: 5) of a murine (musmusculus) variant of R-cadherin preproprotein reported by Hutton et al.in the SWISS-PROT database as Accession No. NP 033997, version NP033997.1, GI:6753376, the relevant disclosure of which is incorporatedherein by reference. SEQ ID NO: 5 includes the amino acid sequenceIDSMSGR (SEQ ID NO: 2) at positions 219-225.

Non-selective peptide antagonists of cadherins including the amino acidsequence His-Ala-Val (HAV) have been reported by Blaschuk et al. in U.S.Pat. No. 6,465,427, U.S. Pat. No. 6,3456,512, U.S. Pat. No. 6,169,071,and U.S. Pat. No. 6,031,072. Blaschuk et al. have reported both linearand cyclic peptide antagonists of cadherins, all of which are capable ofantagonizing a number of types of cadherin molecules indiscriminately.

Selective peptide antagonists of N-cadherin, which comprise the aminoacid sequence Ile-Asn-Pro (INP) have been reported by Williams et al.,Mol. Cell Neurosci., 2000;15(5):456-64. While HAV peptides arenon-specific cadherin antagonists, the INP peptide antagonists reportedby Williams et al. are specific for N-cadherin and do not exhibitsignificant binding to other cadherin molecules such as R-cadherin.

Because of the differential distribution of cell adhesion molecules invarious tissues in the body, there is an ongoing need for antagoniststhat are highly selective for specific cell adhesion molecules, inparticular for antagonists that are selective for R-cadherin. Theselective R-cadherin antagonist peptides of the present inventionfulfill this need.

SUMMARY OF THE INVENTION

An isolated peptide useful as a selective antagonist of mammalianR-cadherin comprises 3 to 30 amino acid residues, three contiguousresidues of the peptide having the amino acid sequence Ile-Xaa-Ser(IXS); wherein Xaa is an amino acid residue selected from the groupconsisting of Asp, Asn, Glu, and Gln. Preferably Xaa is Asp or Asn. Inone preferred embodiment the peptide comprises at least seven amino acidresidues and seven contiguous amino acid residues of the peptide havethe amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6), withXaa being the same as defined above. The present invention also providespharmaceutical compositions comprising the R-cadherin antagonistpeptides in a pharmaceutically acceptable carrier.

In another preferred embodiment the peptide is a cyclic peptide having 3to 10 amino acid residues arranged in a ring, three contiguous residuesof the peptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaais an amino acid residue selected from the group consisting of Asp, Asn,Glu, and Gln. Preferably Xaa is Asp or Asn.

A preferred cyclic peptide has the formula:

-   -   wherein X¹ and X² are independently an amino acid residue or a        plurality of up to 10 amino acid residues linked by pentide        bonds; Y¹ and Y² are independently amino acid residues linked to        one another by a disulfide bond and Xaa is an amino acid residue        selected from the group consisting of Asp, Asn, Glu, and Gln.

A particularly preferred cyclic peptide has the amino acid sequencecyclic Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7); wherein Xaa is an amino acidresidue selected from the group consisting of Asp, Asn, Glu, and Gln,and the peptide ring is formed by a disulfide linkage between the twocysteine residues.

A method of inhibiting R-cadherin mediated cellular adhesion involvescontacting R-cadherin expressing cells with an adhesion inhibitingamount of a selective R-cadherin antagonist peptide of the presentinvention. For example, retinal angiogenesis is inhibited byadministering to a patient suffering from abnormal retinal vascularangiogenesis an angiogenesis inhibiting amount of a R-cadherinantagonist peptide of the present invention. Similarly, targeting oflineage negative hematopoietic stem cells to developing vasculature isinhibited by contacting the stem cells with a vasculature targetinginhibiting amount of a R-cadherin antagonist peptide of the presentinvention. Inhibiting targeting of Lin⁻HSCs, such as endothelialprecursor cells, to developing vasculature is useful for treatingdiseases associated with abnormal vascular development such as agerelated macular degeneration and diabetic retinopathy.

BRIEF DESCRIPTION OF THE DRAWINGS

In the Drawings, FIG. 1 is a schematic representation of cadherinclustering and cadherin modulated cellular adhesion;

FIG. 2 depicts the amino acid sequence of a human variant of R-cadherinpreproprotein (SEQ ID NO: 1), which includes the sequence IDSMSGR (SEQID NO: 2) at residues 222-228;

FIG. 3 depicts the amino acid sequence of a human variant of R-cadherinpreproprotein (SEQ ID NO: 3), which includes the sequence INSMSGR (SEQID NO: 4) at residues 222-228;

FIG. 4 depicts the amino acid sequence of a murine variant of R-cadherinpreproprotein (SEQ ID NO: 5), which includes the sequence IDSMSGR (SEQID NO: 2) at residues 229-225;

FIG. 5(A) illustrates the sequence homology within residues 24-92 ofmurine N-cadherin and various R-cadherins (conserved residues (blue) andnon-conserved (red) residues); note homologies between mammalianR-cadherins from human, mouse and rat, all of which comprise a sequenceIDS or INS at residues 53-55, in contrast to chicken R-cadherin andmouse N-cadherin, which have the sequence IDP and INP, respectively atresidues 53-55; (B) cyclic and linear peptides corresponding to residueswithin this region of murine and human R-cadherin and murine N-cadherinwere synthesized along with the corresponding control peptides: cyclicCIDSC (SEQ ID NO: 8), cyclic CINPC (SEQ ID NO: 9), IDSMSGR (SEQ ID NO:2), IDSASGR (SEQ ID NO: 10), INPASGQ (SEQ ID NO: 11), cyclic CSDIC (SEQID NO: 12), and cyclic CRADC (SEQ ID NO: 13); the partial cadherinsequences listed in FIG. 5(A) are, from top to bottom, murine N-cadherin(SEQ ID NO: 14), murine R-cadherin (SEQ ID NO: 15), rat R-cadherin (SEQID NO: 16), a human R-cadherin (SEQ ID NO: 17), another human R-cadherin(SEQ ID NO: 18), and chicken (gallus gallus) R-cadherin (SEQ ID NO: 19);

FIG. 6(A) depicts photomicrographs demonstrating aggregation of L-cellsexpressing R-cadherin and N-cadherin; (B) is a bar graph of percentaggregation of L-cells mediated by R and N-cadherins in the presence andabsence of calcium;

FIG. 7 demonstrates stable transfection of L-cells with R-cadherin andN-cadherin; (A) R-cadherin immunoblot; (B) N-cadherin immunoblot; (C-E)photomicrographs of stained L-cells demonstrating expression ofR-cadherin (C), and N-cadherin (D), compared to cells expressing neitherR nor N-cadherin (E);

FIG. 8 graphically illustrates selective inhibition of aggregation ofcadherin expressing L-cells by IDS containing peptides, which bind toR-cadherin expressing cells, compared with INP containing peptides,which bind to N-cadherin expressing cells;

FIG. 9 illustrates selective inhibition of mouse retinal vascularizationafter intravitreal injection of cyclic CIDSC (SEQ ID NO: 8), comparedwith cyclic CINPC (SEQ ID NO: 9); (A) depicts photomicrographs of rd/rdmouse retinas at the P2 stage of development; (B) depictsphotomicrographs of rd/rd mouse retinas at the P7 stage of development;(C) is a bar graph of superficial vascularization; (D) is a bar graph ofdeep vascularization;

FIG. 10 illustrates the results of flow cytometry analysis of R-cadherinexpression in hematopoietic stem cells (HSC);

FIG. 11 depicts cross-sectional photomicrographs of rd/rd mouse retinastreated with various peptides of the invention and control peptides; and

FIG. 12 illustrates the blocking of Lin⁻HSC targeting of developingretinal vasculature by the R-cadherin antagonist peptides of theinvention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein and in the appended claims, the term “cyclic peptide”refers to molecules comprising a plurality of amino acids linkedtogether in a chain by peptide linkages, the ends of the chain beingjoined together to form a ring of amino acid residues. The cyclicpeptide can be joined together by a peptide bond, a disulfide linkagebetween two amino acid residues such as cysteine residues, or by anyother suitable linking group. Nonpeptidal linking groups can be anychemical moiety that can react with functional groups at each end of thepeptide chain to form a link therebetween. For example, two ends of apeptide chain can be linked together by a non-protein amino acid such as3-aminobutyric acid or by a disulfide formed from nonpeptidal thiolgroups such as a thioglycolic amide at the amino terminal end and amideformed from 2-aminoethane thiol at the carboxy terminal end, forexample.

As used herein and in the appended claims, the term “pharmaceuticallyacceptable” and grammatical variations thereof, in reference to carriersand other excipients, means that the materials are capable ofadministration to a human patient without the production of undesirablephysiological side effects such as retinal or ocular irritation, nausea,dizziness, blurred or impaired vision, cytotoxicity, and the like.

The term “amino acid” as used herein and in the appended claims refersgenerally to any alpha amino acid. Preferably, the peptides of thepresent invention comprise the 21 amino acids encoded by the geneticcode, although modified amino acid residues can also be included. Theamino acids can be in the L, D, or D,L form. Preferably, the peptides ofthe present invention comprise L-form amino acids. To minimize thelikelihood of proteinase degradation in vivo, the administered peptidesof the present invention can include one or more D-form amino acidresidues.

An isolated peptide, which is a selective antagonist of mammalianR-cadherin comprises 3 to 30 amino acid residues, three contiguousresidues of the peptide having the amino acid sequence Ile-Xaa-Ser. Xaais an amino acid residue selected from the group consisting of Asp, Asn,Glu, and Gln. Preferably Xaa is Asp or Asn. The R-cadherin antagonistpeptide of the present invention can be linear or cyclic.

The selective R-cadherin antagonist peptides of the present inventionmimic the Ile-Asp-Ser and Ile-Asn-Ser sequences found in the EC1 domainof mammalian R-cadherin, but not in other cadherin molecules. Peptidescomprising the Ile-Xaa-Ser sequence can bind to and antagonize mammalianR-cadherin molecules. Xaa preferably is an aspartic acid residue (Asp)or an asparagine residue (Asn), to match the naturally occurringsequences in mammalian R-cadherin molecules. Glutamic acid (Glu) andglutamine (Gln) residues are also suitable for Xaa, due to theirchemical similarity to Asp and Asn, respectively.

In one preferred embodiment, the peptide comprises at least seven aminoacid residues and seven contiguous amino acid residues of the peptidehave the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6).Xaa is an amino acid residue selected from the group consisting of Asp,Asn, Glu, and Gln. Preferably Xaa is Asp or Asn.

In another preferred embodiment the peptide is a cyclic peptide having 3to 10 amino acid residues arranged in a ring, three contiguous residuesof the peptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaais an amino acid residue selected from the group consisting of Asp, Asn,Glu, and Gln, as described above. Preferably Xaa is Asp or Asn.

A preferred cyclic peptide having five amino acids arranged in a ringhas the formula:

-   -   wherein X¹ and X² are independently an amino acid residue or a        plurality of up to 10 amino acid residues linked by peptide        bonds; Y¹ and Y² are independently amino acid residues linked to        one another by a disulfide bond and Xaa is an amino acid residue        selected from the group consisting of Asp, Asn, Glu, and Gln.        Preferably Y¹ and Y² are both cysteine residues linked together        by a disulfide bond (i.e., a cystine residue).

A particularly preferred cyclic peptide has the amino acid sequencecyclo Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7); wherein Xaa is an amino acidresidue selected from the group consisting of Asp, Asn, Glu, and Gln,and the ring is formed by a disulfide linkage between the two cysteineresidues. Preferably Xaa is Asp or Asn.

A method of inhibiting R-cadherin mediated cellular adhesion involvescontacting R-cadherin expressing cells with an adhesion inhibitingamount of a R-cadherin antagonist peptide of the present invention. Thecells can be contacted in vivo with the peptide antagonist byadministering a cellular adhesion inhibiting amount of the antagonist toa mammal suffering from a disease or condition that is treatable byinhibiting R-cadherin mediated cellular adhesion (e.g., retinal diseasescharacterized by abnormal vascular proliferation). For example, a humanpatient suffering from age related macular degeneration or diabeticretinopathy can be beneficially treated with a selective R-cadherinantagonist peptide of the present invention. Preferably the antagonistis administered as a pharmaceutical composition comprising theantagonist and a pharmaceutically acceptable carrier therefor.

For the selective targeting or antagonism of R-cadherin the peptides andcompositions of the present invention may be administered in atherapeutically effective amount parenterally, orally, by inhalation, ortopically in unit dosage form together with pharmaceutically acceptablecarriers, vehicles, and adjuvants. The term “parenteral,” as usedherein, includes intravenous, subcutaneous, intramuscular, intrasternal,intraocular (e.g. intravitreal), and intraperitoneal administration, aswell as administration by infusion techniques.

Any suitable route of administration can be utilized, and thepharmaceutical composition including a selective R-cadherin antagonistpeptide of the present invention is administered in a dose effective forthe intended treatment. Therapeutically effective amounts required totreat a particular medical condition, or inhibit the progress thereof,are readily determined by those skilled in the art using preclinical andclinical studies known in the medical arts.

The term “therapeutically effective amount,” as used herein, refers tothat amount of active ingredient that elicits the biological or medicalresponse of a tissue, system, animal or human, sought by a clinician ora researcher.

The term “inhibit,” as used herein, refers to a slowing, interruption,or stoppage of the medical condition or a biochemical interaction, butdoes not necessarily indicate a total elimination of the condition orcomplete interruption of the interaction. A prolonged survivability of apatient or prolonged reduction in the severity of symptoms, in and ofitself, indicates that the medical condition is beneficially controlled(i.e., inhibited).

The dosage regimens for the present R-cadherin antagonist peptides andcompositions containing the same, are based on several factors such asthe age, weight, sex, and type of medical condition of the patient, theseverity of the condition, the route of administration, and theantagonist activity of the particular peptide antagonist employed. Thedosage regimen may vary depending upon the aforementioned factors.Dosage levels on the order of about 0.01 milligram to about 1000milligrams per kilogram of body weight are useful for inhibiting retinalangiogenesis, for example. Preferred dosage levels are in the range ofabout 0.01 milligram to about 100 milligrams per kilogram of bodyweight.

For administration by injection, a peptide-containing compositionembodying the present invention is formulated with a pharmaceuticallyacceptable carrier such as water, saline, or an aqueous dextrosesolution. For injection, a typical daily dose is about 0.01 milligram toabout 100 milligrams per kilogram of body weight, iniected daily as asingle dose or as multiple doses depending upon the aforementionedfactors.

Pharmaceutical compositions of the present invention comprising aselective R-cadherin antagonist peptide of the invention and apharmaceutically acceptable carrier can also include pharmaceuticallyacceptable excipients. Pharmaceutically acceptable excipients that canbe included in the pharmaceutical compositions of the present inventioninclude, for example, physiologically tolerable surfactants, solvents,buffering agents, preservatives, and the like, which are well known inthe art.

To inhibit retinal angiogenesis, for example, a patient suffering fromabnormal retinal vascular proliferation is administered atherapeutically effective amount of a R-cadherin antagonist peptide ofthe present invention. The administered peptide selectively binds toR-cadherin in the retina, thus disrupting and inhibiting angiogenesistherein. Preferably the peptide antagonist is administered byintravitreal injection.

Targeting of Lin⁻HSCs to developing vasculature is inhibited bycontacting the stem cells with a vasculature targeting inhibiting amountof a selective R-cadherin antagonist peptide of the present invention.Inhibiting targeting of Lin⁻HSCs, such as endothelial precursor cells,to developing vasculature is useful for treating diseases associatedwith abnormal vascular development such as age related maculardegeneration and diabetic retinopathy. Preferably the Lin⁻HSCs arecontacted in vivo by administering the present R-cadherin antagonistpeptides to a mammal, such as a human, suffering from a vascularproliferative disease or condition.

The following non-limiting examples are provided to further illustratethe various aspects of the invention. One of skill in the art willrecognize that modifications of the examples and illustrated embodimentsdisclosed herein can be made without departure from the spirit and scopeof the invention.

EXAMPLE 1 Peptide Synthesis

The peptides of the present invention and various control peptides weresynthesized by The Scripps Research Institute Protein and Nucleic Acidscore facility using the solid phase synthesis method, and were purifiedto the highest grade possible (>95% pure) as analyzed by HPLC analysis.The sequences of the peptides were analyzed by mass spectrometry toensure synthesis of the correct peptides. All peptides were amideblocked at the amino terminus and acetylated at the carboxy terminus.The cyclic peptides were prepared with cysteine residues at the aminoand carboxy terminal ends to create a disulfide tether and form a ringcontaining five amino acid residues. FIG. 5 (B) illustrates the peptidesprepared: cyclic CIDSC, (SEQ ID NO: 9) cyclic CINPC, (SEQ ID NO: 9)IDSMSGR, (SEQ ID NO: 2) IDSASGR, (SEQ ID NO: 10) INPASGQ, (SEQ ID NO:11) cyclic CSDIC, (SEQ ID NO: 12) and cyclic CRADC. (SEQ ID NO: 13)

EXAMPLE 2 L-Cell Transfections

Mouse R-cadherin and N-cadherin plasmids were generous gifts from Dr.Masatoshi Takeichi (Kyoto University, Japan). The plasmids weresub-cloned into pDsRed2 N1 vectors (Clontech) to encode for fusionproteins with Red Fluorescent Protein (RFP) attached to the C-terminalend of the cadherin molecules. L-cells (mouse fibroblast L929 cells,ATCC #CRL-2148) were stably transfected with either R or N-cadherinpDsRed2 N1 using the Calcium Phosphate Transfection System (LifeTechnologies) according to the manufacturer's protocol. After screeningby growth in media supplemented with Geneticin (700 μg/mL G418Geneticin, Gibco BRL), positive clones were selected. Cells wereexamined for expression of RFP, and were tested for cadherin expressionby immunoblotting and immunofluorescence staining. FIG. 6 illustratesthe aggregation of L-cells expressing R and N-cadherins. FIG. 6(A) showsphotomicrographs of R-cadherin (left) and N-cadherin (middle) expressingL-cells aggregating in calcium containing media, compared tonon-transfected L-cells, which did not aggregate. FIG. 6(B) is a bargraph illustrating the percentage of aggregation of the cells shown inFIG. 6(A). N-cadherin and R-cadherin transfected cells trypsinized in abuffer containing about 5 mM calcium chloride (labeled TC) formed largecell clusters, whereas endogenous L-cells showed little aggregation inthe calcium containing buffer. Cells that were trypsinized with EDTA ina calcium free buffer (labeled TE) showed little aggregation, regardlessof whether the cells were transfected with cadherins or not.

EXAMPLE 3 Cell Culture and Immunofuorescence

Transfected or wild type L-cells were grown in Modified Eagles Medium(MEM) supplemented with Earl's Basic Salt Solution, 2 mM Glutamax, 1 mMsodium pyruvate, 0.1 mM non-essential amino acids, and 10% fetal bovineserum. Transfected cell lines were grown in media supplemented withabout 700 μg/mL G418 (all media reagents were from Gibco BRL). Forimmunofluorescence, the cells were grown to about 75% confluency onglass cover slips. The cells were fixed in 4% paraformaldehyde for aboutone-half hour followed by blocking with 5% normal goat serum and 5%fetal bovine serum in 1× phosphate buffered saline (PBS). Goatpolyclonal antibodies against R-cadherin or N-cadherin (Santa Cruz) wereused at 1:200 dilution, and fluorescence was conferred by incubationwith Alexa488 labeled anti-goat IgG secondaries (Molecular Probes).Images were created using a Radiance 200 fluorescence confocalmicroscope (BioRad). For immunoblot analysis, cells were lysed in buffercontaining 1% Triton X-100. About 50 μg of total cell lysate was addedto each lane of an 8% polyacrylamide gel and proteins separated byelectrophoresis. Monoclonal antibodies (1:1000, BD Biosciences) specificfor N-cadherin or R-cadherin were used to visualize the correspondingbands.

FIG. 7(A) shows an immunoblot of native L-cells and L-cells transfectedwith R-cadherin and N-cadherin and stained with R-cadherin antibody.Only the R-cadherin transfected cells exhibited significant levels ofR-cadherin expression. FIG. 7(B) shows an immunoblot of native L-cellsand L-cells transfected with R-cadherin and N-cadherin and stained withN-cadherin antibody. Only the N-cadherin transfected cells exhibitedsignificant levels of N-cadherin expression. FIG. 7(C) is a fluorescencephotomicrograph of R-cadherin expressing cells labeled with fluorescentcadherin antibodies, demonstrating cell surface expression of only theR-cadherin molecules. FIG. 7(D) is a fluorescence photomicrograph ofN-cadherin expressing cells labeled with fluorescent cadherinantibodies, demonstrating cell surface expression of only the N-cadherinmolecules. FIG. 7(E) is a fluorescence photomicrograph of native L-cellsexposed to fluorescent cadherin antibodies, but showing no cell surfaceexpression of cadherin molecules of either type.

EXAMPLE 4 Aggregation Assay

L-Cells were grown to near confluency followed by trypsinization with0.01% trypsin +5mM CaCl₂ and no EDTA (TC) or 0.01% trypsin with 0.1 mMEDTA and no calcium (TE). The cells were collected and washed, followedby resuspension in Hanks buffer solution (HBSS) +1% BSA with (TC) orwithout (TE) 5mM CaCl₂. Cells were incubated at 37° C. in 0.5 mLsolution at 2×10⁵ cells per well of a 24 well plate with rocking atabout 60-70 rpm with varying peptide concentrations. All assays wereperformed in triplicate. The extent of cellular aggregation wasrepresented by the ratio of the total particle number after 2 hours ofincubation (N_(2hr)) to the initial particle number (N₀). Particles werecounted on a hemocytometer using the sum of 8 separate 20 μLcounts/well, before (N₀) and after (N_(t)) incubation. The results areillustrated in FIG. 8.

EXAMPLE 5 Treatment of Mice by Intravitreal Injection of Peptides

Peptides were dissolved in PBS +10% DMSO to a concentration of 10 mM.About 0.5 μL or 1.0 μL of 10 mM peptide solution was injected into thevitreal cavity of 2 day or 7 day-old mice respectively. At P5 or P11,the retinas were dissected as described and the vessels and astrocytesvisualized by immunohistochemistry. Quantification of peripheralvascularization, vascular length, and vascular area during superficialvascular formation was achieved by imaging injected retinas under thesame microscopy settings. Numbers were then generated using LASERPIX®software (BioRad) with non-injected control littermates used forbaseline normalization of the extent of retinal vascularization.Quantification of the effect on deep vascular formation was achieved byfocusing anterior to the normal deep vascular plexus using confocalmicroscopy, and counting the numbers of vessels that had migrated intothe photoreceptor layer. The results are presented in FIG. 9.

EXAMPLE 6 Stem Cell Isolation and Enrichment

Bone-marrow cells were isolated from adult transgenic mice in whichenhanced GFP was fused to the p-actin promoter (ACTbEGFP, the JacksonLaboratory, Bar Harbor, Me.). Monocytes were then collected by densitygradient separation using Histopaque (Sigma) and labeled withbiotin-conjugated lineage panel antibodies (CD45, CD3, Ly-6G, CD11,TER-119, Pharmingen, San Diego, Calif.) for Lin⁻ selection. Lin⁺ andLin⁻ cells were separated using a magnetic separation column (MACS,Miltenyi Biotech, Auborn, Calif.). Since it was determined that CD31⁻cells represent a better control of a non-functional subpopulation ofHSCs as determined by vascular targeting, CD31⁻ cells were isolated fromthe monocytes by MACS sorting using CD31 antibodies and used as anegative control for the functional Lin⁻HSCs. HSCs from wild type micewere analyzed for the expression of R-cadherin by labeling cells withanti-R-cadherin antibodies (sc-6456, Santa Cruz Biotech) and Alexa-488labeled donkey anti-goat secondaries (Molecular Probes), and using aFACS calibur (Beckton Dickinson, Franklin Lakes, N.J.) flow cytometer.The results are presented in FIG. 10.

EXAMPLE 7 HSC Cell Incubations, Injections, and Quantification

Lin⁻HSCs were incubated with 100 nM of R-cadherin blocking antibody(SC-6456, Santa Cruz Biotech), or pre-immune goat IgG in phosphatebuffered saline solution for about 1 hour at about 37° C. prior toinjection. Intravitreal injections into P6 eyes were performed using 0.5μL of HSC solution. Retinas were then examined at P12 by whole mount orsections. Targeting of the Lin⁻HSCs was quantified by counting the totalnumber of stem cells within the retina using eight different fields ofview per retina: left, right, top, and bottom quadrants (¾ distance tothe retina periphery), two intermediate quadrants (¼-½ distance to theperiphery), the injection site, and the optic nerve head region. Thesecells were characterized by their localization to the superficial,intermediate, or deep layers, or by the lack of targeting (cells thatlie at the back of the photoreceptor layer). The number of non-targetedcells within the photoreceptor layer is given as a percentage of thetotal number of stem cells observed. The results are presented in FIG.11 and FIG. 12.

Discussion

The selective R-cadherin antagonist peptides of the present inventionact as peptide mimetics of key recognition motifs of R-cadherin (i.e,the IDS and INS sequences found in mammalian R-cadherins). Without beingbound by theory, the present antagonist peptides likely block theadhesion function of R-cadherin molecules by competitive interactionwith the EC1 domains of R-cadherin molecules on cell surfaces. Thepresent antagonist peptides are useful for the study of molecularfunctions, and for the treatment of cellular adhesion-related diseases,and are generally are more diffusible within a tissue upon in vivoinjection than antibody-based antagonists.

Tissue morphogenesis during the development of most tissues, includingretinal neural tissue, involves the selective binding of cell-celladhesion molecules. This binding selectivity allows similarlydifferentiated cells to organize together, and prevents cell types frominvading incorrect tissue structures. However, despite the extensivestudies on cadherin properties and function, particularly N- andE-cadherins, a general mechanism accounting for cadherin specificity hasnot yet emerged. The present R-cadherin antagonist peptides selectivelyinteract with mammalian R-cadherin molecules without significant bindingto other cadherin classes. These peptides contain the IDS sequence (orits homologs INS, IES, and IQS), which corresponds to a region withincadherin domain EC1, residues 53-55 of SEQ ID NO: 17 and 18, whereimportant interactions within the adhesion interface are reportedlylocated based on structural, mutational, and sequence homology analysis.

Without being bound by theory, the IDS motif is thought to make directcontacts with the VDI sequence from an adjacent cadherin molecule at theadhesion interface. Because residues 53-55 of cadherins appear to berequired for transdimerization, and because unlike otheradhesion-important regions this short sequence of amino acids was notconserved amongst classical cadherin family members, this region acts asa determinant for cadherin specificity. Indeed, cyclic IDS (CIDSC, SEQID NO: 8) selectively inhibits R-cadherin mediated cellular aggregation,while the corresponding N-cadherin counterpart, cyclic INP (CINPC, SEQID NO: 9) selectively inhibits N-cadherin mediated aggregation.N-cadherin and R-cadherin are the most homologous of the cadherin familymembers. In fact, although all cadherins, including R and N-cadherinprefer to interact in a homophillic manner, R- and N-cadherin are theonly two classical cadherin family members where functional heterodimershave been observed. Thus, it is highly unlikely that cyclic IDS andcyclic INP would have distinct functional properties for N andR-cadherin, but overlapping properties for any other cadherin member.These studies demonstrate that the IXS motif (where X is D, N, E, or Q)(i.e. corresponding to R-cadherin residues 53-55, and homologs thereof),plays an important role in mediating homoassociation of R-cadherinmolecules. It is likely, based on the specificity of IXS for R-cadherinand INP for N-cadherin, that correspondingly placed residues in othercadherin family members (e.g., the IER motif of E-cadherin and the IEKmotif of P-cadherin) also impart specificity to the other classicalcadherins as well.

Previous studies have shown that antibodies against R-cadherin disruptretinal vascularization in vivo. Vascularization of the superficialplexus likely was disrupted due to the interruption of R-cadherinmediated guidance cues relayed by astrocytes lying ahead of theendothelial cells. R-cadherin expression is also observed in the regionswhere the deep vascular plexuses are subsequently located, just ahead ofvascular invasion. R-cadherin molecules within these regions are thoughtto guide endothelial cells to the correct vascular plexus, sinceinjection of R-cadherin blocking antibodies causes vessels to bypass thenormal vascularized layers.

Similar vascular phenotypes have now been generated by injection ofcyclic IDS (CIDSC, SEQ ID NO: 8) during both superficial and deepretinal vascularization. Since cyclic IDS selectively disruptedR-cadherin mediated aggregation to a significant extent in vitro (i.e.,without significant disruption of N-cadherin mediated aggregation), itis likely that the in vivo vascular phenotype was generated by highaffinity interactions of cyclic IDS with R-cadherin, as well. Inaddition, injection of cyclic INP (CINPC, SEQ ID NO: 9), which was aneffective inhibitor of N-cadherin mediated aggregation but notR-cadherin aggregation in vitro, did not result in a significant retinalvascular phenotype. Together, these results confirm a specific role forR-cadherin during vascular guidance.

The design of the selective R-cadherin antagonist peptides of thepresent invention was based on structural, biochemical, and mutationalanalysis of various members of the classical cadherin family.Tryptophan-2 is known to be important for cadherin function along withthe HAV sequence at amino acid residues 79-81 of N-cadherin andR-cadherin. In fact, linear and cyclic peptides containing the HAVsequence reportedly block N-cadherin mediated neurite outgrowth invitro. However, these sequences are absolutely conserved across allcadherin molecules and therefore carinot confer specificity of binding.Other residues must also make important contacts within the dimerizationinterface, with some non-conserved residues important for cadherinrecognition. Attention was focused on residues within the amino terminalcadherin repeat (EC1). The majority of contact-important residues werelocalized to three regions, amino acids 35-45, amino acids 53-59, andamino acids 79-86. Residues 53-59, contained the majority of thesecadherin specific residues potentially important in the formation of thedimerization interface. Of these, residues 53-55 were of particularsignificance. Thus, peptide mimetics were designed from this region tooptimize the probability that R-cadherin specific peptides would beproduced. Similar peptides against sequences from mouse N-cadherin, themost closely related cadherin family member, and other control peptideswere designed and used for comparative analysis.

Cadherin Mediated Aggregation

Mouse fibroblast cells (lineage L929), commonly referred to as L-cells,were chosen because they are known to contain no endogenous cadherinexpression. R-cadherin stable transfectants were created and used totest the effects of the designed peptides on R-cadherin mediatedaggregation. N-cadherin stable transfectants were also created and usedto evaluate the peptides for cadherin selectivity, based on theireffects on N-cadherin mediated aggregation. Immunoblot analysis detectedhigh levels of R-cadherin and no N-cadherin expression in R-cadherinclone 8 (R-cad8), while high levels of N-cadherin expression and noR-cadherin expression was found in N-cadherin clone 3 (N-cad3), as shownin FIG. 7. Immunofluorescence confirmed the expression of theappropriate cadherin in these chosen clones. When tested in theaggregation assay, the morphology of the transfected cell lines wasaltered as a result of cadherin transfection. While the parent (i.e.,non-transfected) L-cells remained dissociated as single cell particles,the mouse R-cadherin and N-cadherin transfectants formed large,calcium-dependent (TC buffer), cell clusters due to tight intercellularassociations, as shown in FIG. 6(A). Cadherin mediated aggregationclusters were eliminated by initial trypsinization of the cells withEDTA solution and aggregation in calcium-free buffer (TE buffer), asshown in FIG. 6(B).

Peptide Effects on Cell Aggregation

Peptides were added at varying concentrations to the aggregation wellsto test their effectiveness at blocking cadherin mediated adhesion.Cyclic IDS inhibited R-cadherin mediated aggregation with an IC₅₀ ofaround 300 μM. The linear peptide IDSMSGR (SEQ ID NO: 2), also blockedR-cadherin mediated adhesion. However, its effectiveness (IC₅₀˜900 μM)was about 3 times lower than that of cyclic IDS. As the cyclic peptidesalso proved to be much more soluble and easier to work with than thelinear peptides, further emphasis was focused solely on analysis ofcyclic peptides (FIG. 8(A)). The effects of cyclic IDS were specific forR-cadherin, as little effect on N-cadherin aggregation was observed. Incontrast, the corresponding N-cadherin specific sequence, cyclic INP,inhibited N-cadherin mediated aggregation with an IC₅₀ just below 300 μM(FIG. 8(B)), similar to the effects of cyclic IDS on R-cadherinaggregation. Cyclic INP had little effect on R-cadherin mediatedaggregation.

The other control peptides, cyclic RAD (CRADC, SEQ ID NO: 13) and cyclicSDI (CSDIC, SEQ ID NO: 12) had little effect on either R-cadherin orN-cadherin mediated aggregation. A cyclic HAV peptide (CHAVC, SEQ ID NO:20), already known to be effective at blocking adhesion mediated by anyclassical cadherin molecules, was tested as a comparison. In our assay,cyclic HAV blocked R-cadherin and N-cadherin mediated aggregation withIC_(50S) between 150 and 200 μM. Thus, cyclic IDS and cyclic INPselectively blocked R or N cadherin adhesion respectively, with onlyslightly lower affinities than the non-specific pan cadherin blockingpeptide. Previous studies using antibodies against R-cadherin were shownto disrupt normal retina developmental vascularization. These antibodieswere also effective at disrupting cadherin mediated aggregation in ourassay system with an IC₅₀ of around 10 nM, as shown in FIG. 8(C).

Effects of Peptides on Retinal Vascularization

Peptides were injected into the vitreal cavity of postnatal mouse eyes.When cyclic IDS or cyclic HAV peptides were injected into two-day oldmouse eyes, and the resulting vasculature was examined three days laterat postnatal day 5 (P5), vascular formation was disrupted with resultssimilar to those observed by antibody injections. These retinas werecharacterized as having less extensive peripheral vascularization, andfewer interconnecting vessels within the vascularized regions comparedto normal, non-injected littermate controls. Overall, vascularization ofthe superficial layer was cut in half by R-cadherin blocking peptideswhile retinas with N-cadherin specific cyclic INP injections wererelatively normal (see FIGS. 9(A-C)).

Selective R-cadherin antagonist peptides of the present inventiondisrupted normal vascularization of the deep retinal layers as well.When cyclic IDS peptide was injected at P7, just before vessels of thesuperficial vascular network dive and begin formation of the deepvascular plexus, the resultant P11 vasculature was characterized bynumerous vascular sprouts that had migrated past the normal deepvascular plexus and into the avascular photoreceptor layer. Again, thisis similar to the effects observed previously when R-cadherin antibodieswere injected. In contrast, the deep vascular plexus of eyes injectedwith cyclic INP peptide formed normally, as shown in FIGS. 9(B and D).

R-Cadherin is Expressed by Lin⁻HSCs

Hematopoietic stem cell (HSC) expression of R-cadherin was analyzed todetermine if R-cadherin cell adhesion molecules were expressed at thecell surface of functionally targeting cells. Using flow cytometryanalysis, R-cadherin was expressed at the cell surface of nearly 80% ofthe Lin⁻ subpopulation of HSCs while only 30% of the Lin⁺ cells expressR-cadherin (FIG. 10). Based on the relative fluorescence intensitiesbetween the two cell populations, it is likely that the Lin⁻ cells alsoexpress higher concentrations of R-cadherin at their cell surface, thanthe small portion of R-cadherin positive Lin⁺ cells. Thus, the majorityof cells within the subpopulation that functionally targets the retinalvasculature express R-cadherin while most of the cells from thenon-targeting subpopulation do not. Interestingly, a differentsubpopulation of HSCs that are CD31, CD34, and Mac1 negative and have notargeting function at all, contained even fewer R-cadherin expressingcells.

R-Cadherin Blocking Antibodies and Peptides Disrupt HSC Targeting

To examine the degree to which R-cadherin cell-cell adhesion functionsin targeting of HSCs to the distinct retinal vascular layers, Lin⁻HSCswere blocked with R-cadherin specific, blocking antibodies prior toinjection. Six days after injection, normal Lin⁻HSCs are only foundlocalized to the three vascular layers: 1) the superficial vascularplexus localized within the ganglion cell layer, 2) the deep vascularplexus localized near the outer plexiform layer, and 3) the intermediatelayer localized at the front edge of the inner nuclear layer. FIG. 11(A)shows cross-sections of retinas after injection of normal Lin⁻HSCs(left), Lin⁻HSCs incubated with adhesion blocking R-cadherin antibodies(middle) and Lin⁻HSCs incubated with pre-immune goat IgG (right).

When the Lin⁻HSCs were pre-incubated with anti-R-cadherin antibodiesprior to injection, many of these cells lost their ability to targetcorrectly, while cells pre-incubated with pre-immune IgG functionsimilar to non-blocked HSCs. Targeting to the deep and intermediatevascular layers appears to be especially affected by blocking R-cadherinadhesion as relatively few R-cadherin blocked HSCs were found localizedwithin these regions. The cells localized to the superficial vascularplexus also appeared less organized and were not co-localized with theendogenous vasculature to the same extent as normal Lin⁻HSCs or thosepre-incubated with pre-immune IgG.

Many of the Lin⁻HSCs pre-incubated with R-cadherin antibody, migratedthrough the retina past all three vascular layers, and attachedthemselves to the outer edge of the photoreceptors near the RPE layer.Almost half of the R-cadherin blocked HSCs were found at the outer edgeof the photoreceptor layer (FIG. 11(B)). In comparison, control retinasinjected with HSCs pre-incubated with pre-immune IgG only had 15% of theHSCs mistargeted to this region. A large portion of the mistargetedcells from the control reginas were found near the injection site, andcan likely be attributed to cells that were released subretinally as theneedle was being removed. When the injection site was excluded from thecalculation, the number of mistargeted pre-immune IgG incubated HSCs wasreduced to 10%. Since almost no Lin⁻HSCs are observed within this “extradeep” layer normally, this small percentage of mistargeted control IgGincubated HSCs can likely be attributed to the fact that the pre-immuneIgG was able to bind to about 10% of the Lin⁻cells (FIG. 10). Thesebound IgG molecules may non-specifically prevent normal adhesion simplydue to steric hindrances. However, the difference between the number ofmistargeted cells due to specific R-cadherin blocking, and non-specificIgG blocking, is significant.

FIG. 12(A) shows confocal images through z-planes of the three vascularplexuses and the outer edge of the photoreceptor layer. Normal targetingwithin correct vascular plexuses and along endogenous vessels (red) wasobserved by Lin⁻HSCs (green) blocked with pre-immune goat IgG. BlockingR-cadherin adhesion caused many Lin⁻HSCs to be localized at the outeredge of the photreceptor layer, and cells targeted to the normalvascular plexuses tended to clump together and were not localized alongendogenous (red) vessels. FIG. 12(B) is a bar graph demonstrating thatthe percentage of mistargeted cells relative to the entire population ofHSCs within the retina was significantly greater for the R-cadherinblocked population of Lin⁻HSCs (P values <0.01).

The foregoing description is to be taken as illustrative, but notlimiting. Still other variants within the spirit and scope of thepresent invention will readily present themselves to those skilled inthe art.

1. An isolated peptide which is a selective antagonist of R-cadherin andcomprises 3 to 30 amino acid residues, three contiguous residues of thepeptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaa is anamino acid residue selected from the group consisting of Asp, Asn, Glu,and Gln.
 2. The peptide of claim 1 wherein Xaa is Asp or Asn.
 3. Thepeptide of claim 1 wherein Xaa is Asp.
 4. The peptide of claim 1 whereinXaa is Asn.
 5. The peptide of claim 1 wherein the peptide comprises atleast 7 amino acid residues, seven contiguous amino acid residues of thepeptide having the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQID NO: 6).
 6. The peptide of claim 5 wherein Xaa is Asp or Asn.
 7. Thepeptide of claim 5 wherein Xaa is Asp.
 8. The peptide of claim 5 whereinXaa is Asn.
 9. An isolated peptide consisting of seven amino acidresidues and having the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg(SEQ ID NO: 6); wherein Xaa is an amino acid residue selected from thegroup consisting of Asp, Asn, Glu, and Gln.
 10. A cyclic peptidecomprising 3 to 10 amino acid residues arranged in a ring, threecontiguous residues of the cyclic peptide having the amino acid sequenceIle-Xaa-Ser; wherein Xaa is an amino acid residue selected from thegroup consisting of Asp, Asn, Glu, and Gln.
 11. The cyclic peptide ofclaim 10 wherein Xaa is Asn or Asn.
 12. The cyclic peptide of claim 10wherein Xaa is Asp.
 13. The cyclic peptide of claim 10 wherein Xaa isAsn.
 14. The cyclic peptide of claim 10 wherein the peptide comprises atleast 7 amino acid residues, seven contiguous amino acid residues of thepeptide having the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQID NO: 6).
 15. The cyclic peptide of claim 14 wherein Xaa is Asp or Asn.16. The cyclic peptide of claim 14 wherein Xaa is Asp.
 17. The cyclicpeptide of claim 14 wherein Xaa is Asn.
 18. The cyclic peptide of claim10 represented by the formula:

wherein X¹ and X² are independently an amino acid residue or a pluralityof up to 10 amino acid residues linked by peptide bonds; and Y¹ and Y²are independently amino acid residues linked to one another by adisulfide bond.
 19. The cyclic peptide of claim 18 wherein Y¹ and Y² arecysteine residues linked to one another by a disulfide bond.
 20. Anisolated cyclic peptide having the amino acid sequenceCys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7); wherein Xaa is an amino acid residueselected from the group consisting of Asp, Asn, Glu, and Gln, and thepeptide includes a disulfide linkage between the two Cys residues.
 21. Apharmaceutical composition for inhibiting retinal angiogenesiscomprising an isolated peptide having 3 to 30 amino acid residues, and apharmaceutically acceptable carrier therefor, together with apharmaceutically acceptable excipient, wherein three contiguous residuesof the peptide have the amino acid sequence Ile-Xaa-Ser, and Xaa is anamino acid residue selected from the group consisting of Asp, Asn, Glu,and Gln.
 22. The composition of claim 21 wherein Xaa is Asp or Asn. 23.The composition of claim 21 wherein Xaa is Asp.
 24. The composition ofclaim 21 wherein Xaa is Asn.
 25. The composition of claim 21 wherein thepeptide comprises at least 7 amino acid residues, seven contiguous aminoacid residues of the peptide having the amino acid sequenceIle-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6).
 26. The composition of claim25 wherein Xaa is Asp or Asn.
 27. The composition of claim 25 whereinXaa is Asp.
 28. The composition of claim 25 wherein Xaa is Asn.
 29. Thecomposition of claim 25 wherein the peptide has the amino acid sequenceIle-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6).
 30. A pharmaceuticalcomposition for inhibiting retinal angiogenesis comprising a cyclicpeptide having 3 to 10 amino acid residues arranged in a ring, and apharmaceutically acceptable carrier therefor, together with apharmaceutically acceptable excipient, wherein three contiguous residuesof the cyclic peptide have the amino acid sequence Ile-Xaa-Ser; and Xaais an amino acid residue selected from the group consisting of Asp, Asn,Glu, and Gln.
 31. The composition of claim 30 wherein Xaa is Asp or Asn.32. The composition of claim 30 wherein Xaa is Asp.
 33. The compositionof claim 30 wherein Xaa is Asn.
 34. The composition of claim 30 whereinthe cyclic peptide comprises at least 7 amino acid residues, sevencontiguous amino acid residues of the cyclic peptide having the aminoacid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQ ID NO: 6).
 35. Thecomposition of claim 34 wherein Xaa is Asp or Asn.
 36. The compositionof claim 34 wherein Xaa is Asp.
 37. The composition of claim 34 whereinXaa is Asn.
 38. The composition of claim 34 wherein the cyclic peptideis represented by the formula:

wherein X¹ and X² are independently an amino acid residue or a pluralityof up to 10 amino acid residues linked by peptide bonds; and Y¹ and Y²are independently amino acid residues linked to one another by adisulfide bond.
 39. The composition of claim 38 wherein Y¹ and Y² arecysteine residues linked to one another by a disulfide bond.
 40. Thecomposition of claim 34 wherein the cyclic peptide has the amino acidsequence Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7), and includes a disulfidelinkage between the two Cys residues.
 41. A method of inhibitingR-cadherin mediated cellular adhesion comprising contacting mammaliancells expressing R-cadherin molecules on their cell surface with a celladhesion inhibiting amount of a selective R-cadherin antagonist peptidecomprising 3 to 30 amino acid residues, three contiguous residues of thepeptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaa is anamino acid residue selected from the group consisting of Asp, Asn, Glu,and Gln.
 42. The method of claim 41 wherein the peptide comprises atleast 7 amino acid residues, seven contiguous amino acid residues of thepeptide having the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQID NO: 6).
 43. The method of claim 41 wherein the peptide is a cyclicpeptide comprising 3 to 10 amino acid residues arranged in a ring, threecontiguous residues of the cyclic peptide having the amino acid sequenceIle-Xaa-Ser.
 44. The method of claim 41 wherein the cyclic peptide hasthe amino acid sequence Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7), and includesa disulfide linkage between the two Cys residues.
 45. A method ofinhibiting retinal angiogenesis comprising administering to a patientsuffering from abnormal retinal vascular angiogenesis an angiogenesisinhibiting amount of a selective R-cadherin antagonist peptidecomprising 3 to 30 amino acid residues, three contiguous residues of thepeptide having the amino acid sequence Ile-Xaa-Ser; wherein Xaa is anamino acid residue selected from the group consisting of Asp, Asn, Glu,and Gln.
 46. The method of claim 45 wherein the peptide comprises atleast 7 amino acid residues, seven contiguous amino acid residues of thepeptide having the amino acid sequence Ile-Xaa-Ser-Met-Ser-Gly-Arg (SEQID NO: 6).
 47. The method of claim 45 wherein the peptide is a cyclicpeptide comprising 3 to 10 amino acid residues arranged in a ring, threecontiguous residues of the cyclic peptide having the amino acid sequenceIle-Xaa-Ser.
 48. The method of claim 47 wherein the cyclic peptide hasthe amino acid sequence Cys-Ile-Xaa-Ser-Cys (SEQ ID NO: 7), and includesa disulfide linkage between the two Cys residues.
 49. A method ofinhibiting the targeting of stem cells to developing vasculature fortreating a disease associated with abnormal vascular developmentcomprising contacting mammalian stem cells with a vasculature targetinginhibiting amount of an isolated, selective R-cadherin antagonistpeptide of claim
 1. 50. The method of claim 49 wherein the disease ismacular degeneration or diabetic retinopathy.
 51. The method of claim 49wherein the stem cells are endothelial precursor cells.